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Amyloid biomarkers in Alzheimer’sdiseaseKaj Blennow1,2, Niklas Mattsson1,3,4, Michael Scho ll5,6, Oskar Hansson7,8, andHenrik Zetterberg1,9

1 Clinical Neurochemistry Laboratory, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of

Gothenburg, Molndal, Sweden2 The Torsten Soderberg Professorship at the Royal Swedish Academy of Sciences3 Department of Veterans Affairs Medical Center, Center for Imaging of Neurodegenerative Diseases, San Francisco, CA, USA4 Department of Radiology and Biomedical Imaging, University of California, San Francisco, CA, USA5 Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA6 Department of Clinical Neuroscience and Rehabilitation, University of Gothenburg, Gothenburg, Sweden7 Department of Clinical Sciences, Lund University, Lund, Sweden8 Clinical Memory Research unit, Clinical Sciences, Lund University, Lund, Sweden9 UCL Institute of Neurology, Queen Square, London, UK

Review

Aggregation of amyloid-b (Ab) into oligomers, fibrils,and plaques is central in the molecular pathogenesis ofAlzheimer’s disease (AD), and is the main focus of ADdrug development. Biomarkers to monitor Ab metabo-lism and aggregation directly in patients are importantfor further detailed study of the involvement of Ab indisease pathogenesis and to monitor the biochemicaleffect of drugs targeting Ab in clinical trials. Further-more, if anti-Ab disease-modifying drugs prove to beeffective clinically, amyloid biomarkers will be of specialvalue in the clinic to identify patients with brain amyloiddeposition at risk for progression to AD dementia, toenable initiation of treatment before neurodegenerationis too severe, and to monitor drug effects on Ab metab-olism or pathology to guide dosage. Two types of amy-loid biomarker have been developed: Ab-binding ligandsfor use in positron emission tomography (PET) andassays to measure Ab42 in cerebrospinal fluid (CSF).In this review, we present the rationales behind thesebiomarkers and compare their ability to measure Abplaque load in the brain. We also review possible short-comings and the need of standardization of both bio-markers, as well as their implementation in the clinic.

Amyloid in Alzheimer’s disease‘Amyloid’ is the term used for proteins that are misfoldedinto a cross b-sheet structure and thereby bind dyes suchas Congo Red and Thioflavin T [1]. AD is one of the majoramyloidoses, with two types of amyloid deposited in thebrain: (i) Ab forming aggregates in the form of plaques andcerebrovascular amyloid angiopathy (CAA); and (ii) tauprotein, which forms neurofibrillary tangles, dystrophicneurites, and neuropil threads (reviewed in [2]). When

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� 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tips.2015.03.002

Corresponding author: Blennow, K. ([email protected]).Keywords: Alzheimer’s disease; biomarker; b-amyloid (Ab); cerebrospinal fluid;positron emission tomography (PET).

not specified otherwise, we use the term ‘amyloid’ hereto refer to Ab pathology rather than tau pathology.

Research advances during the past two decades haveresulted in detailed knowledge on disease mechanisms. Ab

is produced by the sequential cleavage of amyloid precur-sor protein (APP) by two enzymes, b-site APP-cleavingenzyme 1 (BACE1), also called b-secretase, and the g-secretase complex (Figure 1). The prevailing hypothesisfor AD pathogenesis is called the amyloid cascade hypoth-esis (Figure 2), posing that Ab aggregation is the initiatingmechanistic event, in which the different stages of aggre-gates, from soluble oligomers to insoluble fibrils in plaques,are believed to impair synaptic function and ultimatelydamage neurons, resulting in chronic neurodegenerationleading to cognitive impairment and finally dementia[3]. AD research advances have also generated a largenumber of drug candidates with potential disease-modify-ing effects. Based on the strong belief in the amyloidcascade hypothesis, researchers have placed an over-whelming focus on molecules targeting Ab productionand aggregation in AD drug development, and most drugcandidates tested aim to inhibit Ab toxicity by reducingfurther Ab aggregation and plaque formation. These drugcandidates include secretase inhibitors to lower Ab pro-duction from APP, Ab aggregation inhibitors to inhibit Ab

oligomerization or fibrillization, as well as active andpassive Ab immunotherapies designed to capture eithersoluble or aggregated Ab, or both, which will be eitherdegraded or cleared from the brain (reviewed in [4]).

Alarmingly, an increasing number of large Phase IIIclinical trials on Ab targeting drugs have reported nobeneficial effects on cognitive symptoms in patients withsporadic AD [5–7]. These discouraging reports have causedincreasing concern in the AD research community that theamyloid cascade hypothesis eventually will be falsified,that is, that Ab aggregation is just a bystander, and notthe cause, of neurodegeneration in AD [8]. A more optimis-tic viewpoint is that there are several logical explanationsfor the trial ‘failures’, including that the trials enrolled

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sAPPβ

β-amyloid

β-CTF (C99)

Full-length APP

671

AICD

770

BACE1

γ-secretase42

TRENDS in Pharmacological Sciences

Figure 1. Generation of b-amyloid (Ab) by metabolism of amyloid precursor protein (APP). APP is a transmembrane protein with a large extracellular N terminus. The Ab

domain is partly embedded in the plasma membrane, with 28 amino acids outside the membrane and 14 amino acids embedded in the membrane. APP is cleaved by b-site

APP-cleaving enzyme 1 (BACE1), also called b-secretase, and a large soluble part (sAPPb) is released. The remaining C-terminal fragment, called b-CTF or C99, is then

cleaved by g-secretase, releasing soluble Ab. g-secretase is an intramembranous protease complex, with four components, the active enzyme presenilin, together with

nicastrin, presenilin enhancer (Pen-2), and anterior pharynx-defective (Aph-1).

Review Trends in Pharmacological Sciences xxx xxxx, Vol. xxx, No. x


patients with AD and dementia, which is probably tooadvanced a stage of the disease to enable this type of drugto show any effect on clinical symptoms, and that trialpatients have been diagnosed based on purely clinicalcriteria, which are too nonspecific; thus, trials will com-prise a cohort with only approximately 80% of enrolledpatients having genuine AD pathology [9]. Both of theseshortcomings call for diagnostic biomarkers to aid clini-cians in making an early and accurate diagnosis. In futureclinical trials on Ab-targeting drugs, amyloid biomarkerswould be especially valuable to confirm that enrolledpatients do have Ab pathology and, thus, the disease forwhich the drug is intended, which would increase thepossibility of identifying a positive clinical effect of thedrug [10]. If drug effects will be seen only in subjects withbiomarker evidence of pathology, such biomarkers wouldalso be useful to guide clinical decisions on whether toprescribe Ab-targeting drugs, once these are available.

Another possible explanation for some of the trial fail-ures is that poor drug candidates have been taken to PhaseII and III clinical trials based on promising, but mislead-ing data from preclinical drug development [2]. It has beencommon in AD drug development to test whether novel Ab

drug candidates reduce the Ab plaque load in AD trans-genic mice and, if so, take the drug into large and expen-sive clinical trials without examining whether targetengagement can be verified in humans. There are numer-ous examples of failed trials (e.g., tarenflurbil and phen-serine), which probably are due to the poor predictivepower of these disease models [2,9]. For this reason, itis becoming increasingly common to apply theragnosticbiomarkers during the early stages of AD drug develop-ment [5,11–13]. In this context, amyloid biomarkersapplied in short small-scale trials to prove target engage-ment in Phase I proof-of-principle studies on healthyvolunteers [9] or Phase II proof-of-concept studies inpatients with AD [11] may be valuable in the selectionof drug candidates and may improve success rates in late-stage clinical trials.

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In contrast to most other neurodegenerative brain dis-orders, a set of biomarkers has been developed for thedifferent pathogenic processes in AD and examined in alarge number of clinical studies. These AD biomarkersinclude magnetic resonance imaging (MRI) of hippocampalor whole-brain atrophy, PET evaluation of glucose metab-olism in cortical neurons and glial cells, CSF assays tomeasure tau protein, reflecting the intensity of the neuro-nal degeneration, and phosphorylated tau, reflecting thepresence of tangles, and the two amyloid biomarkersamyloid PET and CSF Ab42 (reviewed in [14]). In thisreview, we focus on the amyloid biomarkers, which havebeen much examined and reviewed individually, while anobjective head-to-head comparison on their performance tomeasure Ab plaque load or Ab metabolism in the brainis lacking. We also discuss mechanistic differences betweenthe amyloid biomarkers and their implementation inclinical trials and in the clinical routine management ofpatients with cognitive symptoms.

Biomarkers for ADAccording to the National Institutes of Health (NIH) Bio-markers Definitions Working Group, a biomarker is de-fined as ‘a characteristic that is objectively measured andevaluated as an indicator of normal biological processes,pathogenic processes, or pharmacologic responses to atherapeutic intervention’ [15]. While the National CancerInstitute at the NIH defines a biomarker as ‘a biologicalmolecule found in blood, other body fluids, or tissues that isa sign of a normal or abnormal process, or of a condition ordisease’ (http://www.cancer.gov/dictionary?cdrid=45618),the World Health Organization (WHO) has a broaderdefinition of biomarkers, which includes ‘almost any mea-surement reflecting an interaction between a biologicalsystem and a potential hazard, which may be chemical,physical, or biological. The measured response may befunctional and physiological, biochemical at the cellularlevel, or a molecular interaction.’ (http://www.inchem.org/documents/ehc/ehc/ehc155.htm).

http://www.cancer.gov/dictionary?cdrid=45618

http://www.inchem.org/documents/ehc/ehc/ehc155.htm

http://www.inchem.org/documents/ehc/ehc/ehc155.htm

Familial AD Sporadic AD

Gradual decreasein CSF Aβ42

Gradual increasein amyloid PET

ligand reten�on

APP muta�onsAPP gene duplica�on

PSEN muta�ons

Life-long disturbance in Aβ produc�on:– increase in total Aβ

– absolute or rela�ve increase in Aβ42– Aβ pep�des with increased tendency for aggrega�on

Progressive cogni�ve dysfunc�on and demen�a

Major effects: aging, APOE ε4 alleleMinor effects: other genes, environmental risk factors

Unknown factors

Failure of Aβ clearance withgradually increasing Aβ levels in brain

Microglial and astrocy�c ac�va�on,inflammatory response, and

oxida�ve stress

Impaired LTP withsynap�c dysfunc�on

Synap�c and neuronal degenera�onwith failure in neurotransmission

Tau pathology

Further aggrega�on resul�ng in fibrillarAβ deposits, with tau-posi�ve dystrophic neurites

and glial response as neuri�c plaques

Aggrega�on of Aβ into diffuse plaques

Conforma�onal change in A βwith forma�on of soluble oligomers

?


Figure 2. The amyloid cascade hypothesis. This is the lead hypothesis for Alzheimer’s disease (AD) pathogenesis, which posits that the central event is an imbalance

between b-amyloid (Ab) production and clearance. In familial AD, genetic alterations cause a life-long disturbance in Ab production or generate Ab peptides that are more

prone to aggregation. In sporadic AD, advanced age and possession of the apolipoprotein E (ApoE) e4 allele have major effects on the risk for developing AD. The common

denominator in the pathogenesis is a conformational change in Ab, which makes it prone to aggregation, with the initial formation of soluble oligomers, followed by larger

fibrils that accumulate into diffuse plaques and, at a later stage, neuritic plaques. Cognitive impairment is believed to be due to Ab oligomers inhibiting hippocampal long-

term potentiation and impaired synaptic function, as well as an inflammatory response, oxidative stress, and synaptic and neuronal degeneration with neurotransmitter

deficits. Tau pathology with tangle formation is regarded a downstream event that contributes to cognitive symptoms. Adapted from [3] with minor modifications,

including the relation between the cascade of pathogenic events and the amyloid biomarkers. Abbreviations: APP, amyloid precursor protein; CSF, cerebrospinal fluid; LTP,

long-term potentiation; PET, positron emission tomography; PSEN, presenilin.



In 1998, the Ronald and Nancy Reagan Research Insti-tute of the Alzheimer’s Association and the NationalInstitute of Aging Working Group set up criteria for anideal AD diagnostic biomarker. These recommendationsstated that the biomarker should be: able to detect afundamental feature of Alzheimer’s neuropathology, vali-dated in neuropathologically confirmed AD cases, precise(able to detect AD early in its course and distinguish itfrom other dementias), reliable, non-invasive, simple toperform, and inexpensive [16]. It may be argued that thereare several clinically useful biomarkers that do not detecta fundamental feature of disease pathology; for example,prostate-specific antigen (PSA) is an excellent biomarkerfor prostate cancer even if not involved in pathophysiology.Nevertheless, as becomes clear below, several of the bio-markers for Ab pathology fulfill most, if not all, of theserequirements.

Amyloid biomarkers in ADThere are two principal AD amyloid biomarkers: amyloidPET, which measures the amount of Ab aggregates in thebrain parenchyma, and biochemical analyses of Ab speciesand APP-processing products in CSF. Several peptides,proteins, and enzymes involved in the amyloidogenic APP-processing can be measured in CSF. The latter includesdifferentially truncated species of Ab, including the longerAb17 up to Ab42 species generated by b- and g-secretasecleavage of APP, and the shorter Ab14 to Ab16 speciesgenerated by b- and a-secretase cleavage [17], soluble b-secretase cleaved APP (sAPPb) [18], and Ab oligomers[19,20]. Except for CSF Ab42, these markers are less wellestablished or show little or no association with AD incross-sectional or longitudinal biomarker studies, butmay still be useful when assessing target engagementfor some of the drug candidates that are being evaluated

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as potential anti-Ab drugs; see, for example, [13]. Here, wefocus on the first biomarker class, but it is important toremember the additional information that can be gainedfrom the second biomarker class in drug development, and,in the future, possibly also to facilitate accurate dose-finding in individual patients.

CSF Ab42

The first report showing that Ab is secreted to the CSFcame in 1992 [21], which made measurement of Ab inCSF an important candidate biomarker for AD. Disap-pointingly, initial reports on CSF Ab showed no clearchange in AD [22,23]. These initial reports were basedon enzyme-linked immunosorbent assays (ELISA) meth-ods that did not discriminate between truncated Ab iso-forms, that is, the ‘total’ CSF level of Ab was measured.The discovery that the 42 amino acid form of Ab, Ab42, isprone to aggregate and the earliest Ab species deposited inplaques [24,25] set focus on immunoassays specific forAb42.

The first paper using an ELISA method specific for Ab42was published in 1995 and showed a marked reduction inCSF in patients with AD [26]. This finding has beenreproduced in numerous studies (reviewed in [27]) andusing many different assay formats, including ELISA[28], Luminex xMAP technology [29], electrochemilumi-nescence immunoassay [18], urea-based SDS-PAGE com-bined with Western blot [30], and antibody-free single-reaction monitoring (SRM) mass spectrometry [31]. InAD dementia, the CSF level of Ab42 is typically decreasedto approximately 50% of the levels in age-matched cogni-tively normal individuals [32].

CSF contains many different Ab isoforms, of whichAb40 is around ten times more abundant than Ab42[33]. CSF levels of Ab40 are unchanged in AD, but thereis a reduction in the CSF Ab42:Ab40 ratio in AD that ismore marked than the reduction in Ab42 alone [34–37]. The CSF Ab42:Ab40 ratio has been suggested tofurther improve diagnostic accuracy, because it can bal-ance interindividual variations in total Ab production(of all Ab isoforms); some non-AD cases that are ‘lowproducers’ may have false positive Ab42 tests (just abovethe cut-off) and some AD cases that are ‘high producers’may have false negative Ab42 tests, while this will becompensated for by the Ab42:Ab40 ratio [35,37].

Amyloid PET

The first study on amyloid PET used an11C-labeled modifiedderivative of the amyloid-binding histological dye Thiofla-vin-T called Pittsburgh Compound-B (PiB) and showedincreased ligand retention in cortical brain regions in ADdementia cases as compared with controls, using cerebellumas a reference region [38]. Increased retention of PiB incortical brain regions in AD has since been verified in manyscientific reports (for review see [39]). The change in globalcortical PiB in cases with AD dementia as compared withcognitively normal older individuals is typically a 50–70%increased retention (range 20–80%) [40].

The short half-life of 11C hinders the use of 11C-PiBoutside expert research centers, unless there is access toan on-site cyclotron and radiochemistry expertise. Thus,

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efforts were initiated to make 18F-labeled tracers, andcurrently four such amyloid ligands have been developed,which have a half-life of approximately 110 min; thisenables centralized production and regional distributionto centers having a PET camera. These ligands include18F-florbetapir (Amyvid), also called AV-45 [41], 18F-flute-metamol (Vizamyl), 30F-PiB or GE-067) [42], 18F-florbeta-ben (Neuraseq), also called BAY94-9172 or AV-1 [43], and18F-NAV4694, formerly known as AZD4694 [44]. Usingthese amyloid tracers, a 40–70% increase in cortical ligandretention is found, but some have higher nonspecific whitematter binding than 11C-PiB, while others show lowercortical binding in patients with AD [44].

Relation to amyloid metabolism and pathology foramyloid biomarkersPiB and other amyloid ligands were developed to bind toaggregated Ab. Binding of the PiB ligand to b-sheet-folded Ab has also been shown in several post-mortemstudies, with preference for compact plaques and vascu-lar deposits called CAA, while diffuse plaques are lessprominently labeled and amorphous plaques comprisingloosely aggregated Ab containing little b–sheet structuredo not bind PiB [45–47]. Furthermore, PiB does not bindaggregated Ab in cases with the arctic APP gene (E693G)mutation [48]. By contrast, human subjects with CAAmay have positive PiB amyloid PET scans, even in theabsence of amyloid plaques [49] and even if the distribu-tion of PiB binding differs between patients with clinical-ly diagnosed AD and CAA [50]. Several studies have alsoshown good agreement between 18F-florbetapir amyloidPET and post-mortem Ab pathology evaluated in hospicepatients near the end of their lives [51,52] and between18F-flutemetamol amyloid PET imaging and cortical fi-brillar Ab pathology in biopsies from living patients withhydrocephalus [53,54].

By contrast, it was less obvious how the reduced CSFlevel of Ab42 would be interpreted. Possible explanationsrange from decreased Ab production, decreased Ab clear-ance (active export from the brain to the blood), aggrega-tion and deposition of Ab in brain tissue, with loweramounts diffusing to the extracellular space and CSF,increased proteolytic degradation of Ab, and possiblymore. The first indication that the lowering of CSFAb42 is due to aggregation of the peptide in the braincame in 2003 in a study showing that low levels of Ab42 inpost-mortem ventricular CSF showed an inverse correla-tion with plaque load in cortical regions [55]. This findinghas been replicated in several studies [56,57]. Furthersupport came from a study in 2006 showing an inverserelation between CSF Ab42 and PiB binding in corticalbrain regions [58], a finding that has been replicated inmany successive papers, as reviewed below. Taken togeth-er, these data indicate that the reduced CSF level of Ab42in patients with AD is due to Ab deposition and, thus,reflects plaque (or fibrillar Ab) load in the brain. However,CSF Ab levels may change dynamically in response topharmacological interventions. For example, a transientlowering of CSF Ab42 is found in response to reduced Ab

production following BACE1 inhibitor treatment, also inhealthy volunteers [13].



Concordance between amyloid biomarkersAfter the first study in 2006 showing an inverse correlationbetween global cortical amyloid PET ligand retention andCSF Ab42 levels [58], several studies confirmed this asso-ciation [59–68]. These ten publications included more than1000 patients and controls who underwent both amyloidPET and CSF Ab42 examinations (Table 1). Most subjects(88%) had concordant amyloid biomarker results, witheither negative or positive amyloid PET scans and CSFAb42 levels (Table 1). A few had discordant amyloid bio-markers results, with either positive (low) CSF Ab42 butnormal amyloid PET (6.6%), or normal CSF Ab42 levelsbut positive amyloid PET scans (5.4%).

Ab plaques in the AD brain comprise several N-termi-nally truncated species, such as Ab3–42, Ab4–42, andpyroglutamate derivatives of Ab (e.g., pGluAb3–42)[69]. These Ab species are not captured by the most com-monly used immunoassay for CSF Ab42, which measuresAb1-42 (i.e., peptides that have the first amino acid pre-served) [28,29]. However, the correlation between amyloidPET positivity and low CSF Ab42 is similar for AbX–42and for Ab1–42 [64]. Thus, amyloid PET correlates withthe total amount of the aggregation-prone Ab42 peptide inCSF. By contrast, no correlations are found for Ab speciesother than Ab42, such as Ab40 or Ab38 [60,64]. These datafurther underline that the correlation reflects the deposi-tion of the aggregation-prone Ab42 peptide in the brain,and not a general change in the metabolism of Ab.

In addition to the amyloid biomarkers CSF Ab42 andamyloid PET, the tau biomarkers CSF T-tau and P-tau alsoshow marked changes in patients with AD dementia andprodromal AD as compared with control populations[70]. Thus, in studies examining mixed AD and controlpopulations, significant correlations are to be expected forall parameters that show a marked difference betweenpatients and controls. Indeed, some studies have shown

Table 1. Agreement between CSF Ab and amyloid PETa,b

PET ligand CSF assay Cases

Florbetapir Ab1-42 (Luminex) Total = 374 (103 normal, 187 ear

62 late MCI, 22 AD)

Flutemetamol Ab1-42 (ELISA) Total original cohort = 118

Total validation cohort = 38c

PiB Ab1-42 (ELISA) Total = 50 (43 CDR = 0, 4 CDR = 0

3 CDR = 1)

Ab1-42 (ELISA) Total = 16 (6 MCI converters,

11 nonconverters)

Ab1-42 (ELISA) Total = 189 (189 controls with CD

Ab1-42 (Luminex) Total = 55 (11 controls, 34 MCI, 1

Ab1-42 (ELISA) Total = 37 (10 controls, 12 MCI, 1

Ab1-42 (ELISA) Total = 10 (10 probable AD)

Ab1-42 (Luminex) Total = 41 (11 controls, 34 MCI, 1

Ab1-42 (ELISA) Total = 136 (64 AD, 34 non-AD, 2

16 controls)

Total 1064

aAbbreviations: AD, Alzheimer’s disease; CDR, clinical dementia rating; CSF, cerebros

bCSF–, normal (= high) CSF Ab42 levels; CSF+ low (below the cut-off in the study) CSF Ab

off in the study) amyloid ligand retention.

cThe cut-off for both amyloid PET and CSF Ab1-42 for the validation cohort was estab

dIncludes the cohort reported in [58].

that patients who are amyloid PET positive have higherCSF T-tau and P-tau levels, but the correlations with CSFT-tau and P-tau are weaker than the strong inverse corre-lation found between amyloid ligand retention and CSFAb42 [60,62,63,67], and do not reach statistical signifi-cance in smaller patient series [58,61,64]. These findingsindicate that the relation between CSF Ab42 and amyloidPET is specifically related to Ab42 deposition in the brain,and not merely to the neurodegenerative disease process inthe AD brain.

Characteristics of discordant casesThe existence of cases that are discordant for CSF and PETamyloid biomarkers has been a subject for discussion,especially the group with low CSF Ab42 but normal amy-loid PET. Early case reports suggested that CSF Ab42decreases before brain Ab accumulation is great enough toenable detection by PET, or that early diffuse Ab deposits,which bind amyloid ligands poorly, occur before fibrillarplaques [71]. A large study comparing amyloid PET andCSF biomarkers and containing a large enough number ofdiscordant cases to allow subanalyses in these groups,showed that discordance (mainly isolated CSF Ab positiv-ity) was clearly dependent on disease stage, and found 21%of cognitively normal older subjects, but only 6% of ADdementia cases, with intermediate frequencies in patientswith mild cognitive impairment (MCI) [72]. These findingsclearly show that CSF-positive/PET-negative biomarkerdiscordance is not driven by technical or methodologicalvariations. Furthermore, CSF Ab42 was more stronglyrelated to possession of the apolipoprotein E (APOE) e4allele than was amyloid PET, while amyloid PET amyloidwas more strongly related to CSF tau and cognitive declinethan was CSF Ab42 [72]. Taken together, these findingsindicate that CSF-positive/PET-negative biomarker mis-matches may be due to CSF Ab42 being more sensitive in

Concordant

CSF–/PET–

or CSF+/PET+

Discordant

CSF+/PET–

Discordant

CSF–/PET+

Refs

ly MCI, 322 (86.1%) 21 (5.6%) 31 (8.3%) [67]

109 (92.4%) 6 (5.1%) 3 (2.5%) [68]

37 (97.4%) 0 1 (2.6%)

.5, 50 (100%) 0 0 [59]d

16 (100%) 0 0 [61]

R = 0) 157 (83%) 28 (15%) 4 (2%) [60]

0 AD) 50 (91%) 2 (4%) 3 (5%) [62]

5 AD) 31 (84%) 6 (16%) 0 [63]

10 (100%) 0 0 [64]

0 AD) 41 (100%) 0 0 [65]

2 MCI, 114 (84%) 7 (5%) 15 (11%) [66]

937 (88.0%) 70 (6.6%) 57 (5.4%)

pinal fluid; MCI, mild cognitive impairment.

42 levels; PET–normal (= low) amyloid ligand retention; PET+, high (above the cut-

lished in the original cohort.

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the early stages of AD, while PET amyloid may still changedynamically during later stages of disease. This interpre-tation is corroborated by findings in a recent study show-ing the cortical flutemetamol retention levels correlatewith disease stage in patients with MCI, while CSFAb42 levels do not [68]. Large studies with longitudinaldata sampling are needed to better understand the tem-poral relation between CSF Ab42 and amyloid PET.

Global or regional amyloid PET versus CSF Ab42In many studies comparing PET and CSF as amyloidbiomarkers, the uptake of the amyloid PET tracer inregional neocortical standardized uptake value ratio(SUVR) is averaged into a single composite SUVR, andnormalized to the uptake in a reference region, most oftenthe cerebellum (Table 2). This approach is feasible becausethe correlation between low CSF Ab42 levels and highretention of amyloid PET tracer is high in almost allcortical regions, with similar correlation coefficients forglobal (or composite) PET tracer retention as for selectedregional cortical (frontal, temporal, posterior cingulate, orparietal) retention [63]. This was verified in a study com-paring flutemetamol amyloid PET and CSF Ab42, showingthat the best correlation is found in brain regions with thehighest ligand retention, suggesting that CSF Ab42 levelsreflect total cortical Ab deposition [68]. In agreement, arecent study showed that, due to the high correlationsbetween regional and global cortical PiB binding potential,many cortical brain regions are comparably useful forclassifying a scan as positive or negative, although withdifferent thresholds [73].

Table 2. Methods and cut-offs for CSF b-amyloid and amyloid PE

CSF b-amyloid (Ab42) Amyloid PET

CSF assay Cut-off for CSF positive PET ligand Cortical and referenc

ELISAb <457 pg/mL 11C PiB Prefrontal, precuneus

gyrus rectus; referen

<500 pg/mL 11C PiB Prefrontal, precuneus

gyrus rectus; referen

<650 pg/md 11C PiB Frontal (mean of orb

superior), parietal, tem

and medial inferior),

(entorhinal and hippo

cingulated; reference

<550 pg/md 11C PiB Posterior cingulum; r

<450 pg/mL 11C PiB Frontal, parietal, tem

cingulum; reference =

<550 pg/mL 11C PiB Frontal, parietal, tem

reference = cerebellu

<647 pg/mL Flutemetamol Reference = cerebellu

Luminexc <192 pg/mL 11C PiB Prefrontal, posterior

lateral temporal, pari

cingulate cortex; refe

cerebellum

<192 pg/mL 11C PiB Prefrontal, orbitofron

anterior cingulate, an

precuneus

<192 pg/mL Florbetapir Reference = cerebellu

aAbbreviations: BPND, nondisplaceable binding potential; MCBP, mean cortical bindin

bInnotest b-amyloid(1-42) (Fujirebio).

cLuminex assay is Alzbio3 (Fujirebio).

dCut-off based from optimal cut-off presented in graph.

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Furthermore, a large study comparing the diagnosticperformance of CSF Ab42 and florbetapir PET in theADNI-2 cohort found similar diagnostic accuracies forthe amyloid biomarkers, and also when comparing CSFAb42 with either global or regional (temporal, frontal,parietal, and cingulate) amyloid PET measurements[74]. These studies do not support that measurement ofcortical amyloid ligand retention in a certain region, suchas posterior cingulate cortex, results in superior diagnosticaccuracy for AD.

Influence of methodology and the need ofstandardization of amyloid biomarkersEven if the amyloid biomarkers (CSF Ab42 and amyloidPET) are highly correlated, there are also differencesdepending on the methodology for quantification ofAb42 in CSF and for evaluation of PET scans. In thestudies evaluating concordance, CSF Ab42 has been ana-lyzed using either ELISA [28] or Luminex xMAP [29]methodologies (Table 2). It is known that the two techni-ques give different absolute levels for CSF Ab42, but showa tight linear correlation [29]. Thus, these methods givedifferent cut-offs for CSF amyloid positivity, approximate-ly 450–650 pg/mL in the papers using the ELISA (cut-offsdefined in different ways), while a cut-off at 192 pg/mL hasbeen used in all papers using the Luminex xMAP tech-nique (Table 2). The 192 pg/mL cut-off for the Luminexassay was originally defined as the point maximizing thediagnostic accuracy in a cohort of autopsy-confirmed ADcases (where pre-mortem CSF was taken several yearsbefore autopsy) and age-matched living healthy controls

Ta

e regions Method for

evaluation

Cut-off for

PET positive

Refs

, lateral temporal and

ce = cerebellum

MCBP (BPND) >0.2 [59]

, lateral temporal, and

ce = cerebellum

MCBP (BPND) >0.18 [60]

ital, medial inferior, and

poral (mean of superior

medial temporal

campus) and posterior

= cerebellum

Global cortical PBND >0.50d [63]

eference = cerebellum ROI/ref PiB retention >1.6d [61]

poral, and posterior

cerebellum

Mean cortical PiB

retention

>1.6 [64]

poral, and occipital;

m

BPND, visually score Positive [66]

m Mean cortical SUVR >1.42 [68]

cingulate/precuneus,

etal, anterior, and

rence = pons and

Mean cortical SUVR >1.465 [62]

tal, parietal, temporal,

d posterior cingulate/

Global cortical PiB

retention

>1.5 [65]

m Mean cortical retention >1.11 [67]

g potential; PBND, normalized ligand binding potential.



[75]. As shown in Table 2, the association between CSFAb42 and amyloid PET is high for both analytical CSFtechniques (Table 2).

In addition to the problem that absolute levels for CSFAb42 vary depending on the analytical technique used(Table 2), there are also differences in absolute levels be-tween laboratories, even if the same assay format is used[76]. In addition, ELISA-based CSF assays are known toshow batch-to-batch variations for the kits [76]. Standardi-zation efforts are ongoing within the Alzheimer’s Associa-tion Global Biomarkers Consortium (GBSC) and theInternational Federation of Clinical Chemistry WorkingGroup for CSF proteins (IFCC WG-CSF), with the aim ofstandardizing both pre-analytical and laboratory proce-dures and to harmonize levels between assay formats[77]. Protocols for standardization of procedures for CSFcollection and sample handling and shipment have beenproposed, together with laboratory procedures for run ac-ceptance and batch bridging to maintain long-term stabilityof ELISA-based CSF biomarkers [10,68]. Within the IFCC-WG-CSF, SRM mass spectrometry-based reference mea-surement procedures (RMP), that is, ‘gold standard’ meth-ods for CSF Ab42, have been published [31,78], and projectsto develop RMPs for CSF tau are ongoing. These methods forabsolute quantification of CSF Ab42 will be used to set theexact levels on a certified reference material (CRM), that is,aliquoted CSF pools with three levels (high, medium, or low)of Ab42 will be used to normalize levels between assayformats [77,79]. Between-laboratory and longitudinal(batch-related) variations are monitored in the Alzheimer’sAssociation Quality Control (QC) program for CSF biomark-ers [10,80]. These standardization efforts have also stimu-lated biotech companies to develop upgraded and validatedversions of assays and to establish CSF biomarker assays onfully automated laboratory analyzers. These developmentswill pave the way for uniform cut-off levels for CSF biomark-ers and a more general use of these diagnostic tools.

In studies evaluating CSF and PET concordance, threedifferent amyloid ligands have been used: PiB, florbetapir,and flutemetamol. Similar concordance rates have beenfound in the eight studies using PiB (84%) as comparedwith florbetapir (86%) and flutemetamol (93%). Further-more, there are differences in the general design of amyloidPET examinations, such as scan duration, type of scan(dynamic/static), camera settings, and methodology usedfor quantification of amyloid ligand binding (Table 2). Acommon measure of amyloid PET ligand retention is thesemiquantitative SUVR, which represents the measuredradioactivity in a volume of interest (VOI) at a certain timepoint, or for a certain time window, of scanning in relationto the injected dose and the individual’s body weight [81],The SUVR is created by normalizing the SUV in a certaintarget VOI to the SUV in a reference region that ideallyshows no or little specific binding (SUVVOI/SUVREF). Thismethod does not require time-consuming dynamic PETscanning or blood sampling for the modeling of ligandkinetics, which renders it a simple and practical tool alsofor clinical settings. Among the studies comparing 11C-PiBamyloid PET and CSF Ab42, only one used mean corticalSUVR, with a cut-off value for a ‘positive’ amyloid PETscan of 1.45 [62]. Another commonly used measure is the

binding potential (nondisplaceable, BPND), which is gener-ally defined as the ratio of receptor density to the dissocia-tion constant (Bmax/KD) [82]. The three studies that usedBPND when comparing amyloid PET with CSF Ab42 hadcut-offs for a ‘positive’ amyloid PET scan from 0.18–0.20[59,60] to 0.50 [63] (Table 2). Lastly, three studies used atechnique in which ligand uptake (raw radioactivity) incortical regions was divided by ligand uptake in the refer-ence region. This measure was called ROI/ref PiB retention[61], mean cortical PiB retention [64], and global corticalPiB retention [65], and had cut-offs for a ‘positive’ amyloidPET scan from 1.5 to 1.6 (Table 2).

The definition of the reference region used to calculateSUVR or BPND measures also differs between studies, withcerebellar gray matter, whole cerebellum, brain stem, andthe pons used as reference regions. The choice of referenceregion may be optimized for different settings. For example,the pons is the preferable reference region in patients withautosomal dominant AD, because these patients may haveamyloid accumulation in the cerebellum [83,84]. Further-more, variability may occur not only when outlining thereference region, but also when defining the VOIs that aresummarized and averaged to create a composite VOI whencalculating MCBP or global SUVR. These VOIs can bedrawn manually, using different protocols, or using auto-mated, often MRI segmentation-based methods. Taken to-gether, the fact that values for tracer binding or retentionvary between tracers, depending on which technique used tocalculate uptake, and likely between PET scanners andcenters, have made uniform cut-off values difficult to estab-lish. The lack of generally accepted protocol for acquisition,processing, and analysis of amyloid PET data also compli-cates comparability between studies. Thus, there is a needfor standardization efforts for amyloid PET biomarkers.Importantly, an international working group recently pro-posed a method for standardizing the quantitative analysisof amyloid PET images across different tracers to enablebetter comparability of amyloid PET data in the context ofcross-center, multicenter, and longitudinal studies, as wellas the definition of cut-offs [85]. According to that proposal,the original PET data are transformed into so-called ‘Cen-tiloids’ (CL), the CL-scale comprising units where 0 repre-sents uptake in an amyloid-negative brain and 100 the brainof a patient with typical mild-moderate AD. Awaiting this,visual evaluation of scans in a binary fashion as amyloid‘positive’ or ‘negative’ has been accepted as the standard,and this has been validated by post-mortem assessment ofAD pathology [52].

Amyloid biomarkers in AD and other brain disordersBoth CSF Ab42 and amyloid PET have in numerousstudies been shown to have a high diagnostic performancefor AD. Approximately 85–90% of clinically diagnosedcases with AD dementia have a positive amyloid PET scan(reviewed in [86]). Approximately 90% of PiB-positive MCIcases progress to AD dementia during clinical follow-up,while most PiB negative cases show stable cognition[61,87,88]. Similarly, most clinically diagnosed AD casesshow low CSF levels of Ab42 [32]. Furthermore, clinicalfollow-up studies have shown that more than 90% of thoseprogressing to AD dementia have low CSF levels of Ab42

7



at baseline [28], while stable MCI cases have normal CSFAb42 [89], findings that have been verified in several largeclinical multicenter studies [75,76,90].

The availability of two amyloid biomarker modalitiesprovides a solid ground to understand the specificity andmechanisms for biomarker results found in brain disordersother than AD. An overview of changes in the amyloidbiomarkers in other disorders than AD is given below.

Other clinical entities with amyloid pathology

Dementia with Lewy bodies (DLB) is characterized bycortical and subcortical Lewy bodies comprising aggregat-ed a-synuclein together with preferentially diffuse corticalamyloid (Ab) plaques [91,92]. PET studies in DLB showhigh cortical PiB retention in most patients with DLB [93],to a degree similar to that found in AD [94]. Studies on CSFAb42 levels have consistently also shown decreased levelsin DLB [95–97]. Some studies have examined whether itmay be possible to differentiate other neurodegenerativedisorders with b-amyloid pathology based on the regionalcortical amyloid PET pattern. Amyloid PET-positive DLBcases show a cortical PiB pattern that is comparable withthat found in patients with AD [96]. The pattern of amyloidPET signal is similar between the different clinical phe-notypes of AD (early-onset AD, logopenic variant primaryprogressive aphasia, and posterior cortical atrophy) [98],and CSF Ab42 levels are reduced independent of pheno-type [99–101]. As mentioned above, cases with CAA havepositive PiB amyloid PET scans, even in the absence ofamyloid plaques [49].

Creutzfeldt-Jakob disease

Studies on CSF Ab42 in Creutzfeldt-Jakob disease (CJD)show contradictory results. Some papers have reportedreduced CSF Ab42 levels [57,102], and it has been debat-ed whether this would indicate that CSF Ab42 may dropdue to reasons other than Ab deposition in the brain, insome neurodegenerative disorders. However, other CSFstudies found no change Ab42 levels [103,104], and amy-loid PET studies on CJD show no cortical PiB retention,with scans indistinguishable from control subjects[105,106], although few cases have been examined inthese studies.

Infections and inflammatory brain disorders

Some reports have shown reduced CSF Ab42 levels ininfectious and inflammatory central nervous system(CNS) disorders. Patients with acute bacterial meningitisshow a marked reduction in CSF Ab42, with normalizationafter proper antibiotic treatment and clinical recovery,while no change is found in cases with viral meningitis[107,108]. In bacterial meningitis, there is a breakdown ofthe blood–brain barrier with high protein levels in CSF,but this was shown not to interfere with the assay for Ab42measurement [107,108]. Instead, the reduction in CSFAb42 may be due to Ab degradation by proteases releasedin response to the acute inflammatory process [109].

Furthermore, some studies report low CSF Ab42, oftenaccompanied by low CSF levels of sAPP, in inflammatoryCNS disorders such as HIV dementia [107,110], systemiclupus erythematosus (SLE) with CNS engagement [111],

8

Lyme neuroborreliosis (LNB) [109], and multiple sclerosis(MS) [112–114]. These findings suggest that CNS inflam-mation results in a general inhibition of APP metabolismwith lower production of Ab. This hypothesis is supportedby the finding that the reduction in CSF Ab is related to adecrease in BACE1 [112], and that the reduction in bothsAPP and Ab species is normalized by treatment ofpatients with MS patients with natalizumab, which inhi-bits transmission of immune cells into the CNS [114], andafter treatment of LNB with doxycycline [109]. However,other studies found normal CSF Ab42 in HIV dementia[115], SLE with CNS engagement [112], LNB [107], andMS [116,117]. Thus, further studies are needed on CSFAb42 in inflammatory CNS disorders to resolve whetheran inflammatory process inhibits APP metabolism and Ab

generation.

Brain trauma

Diffuse axonal injury (DAI) is the central neuropathologi-cal change in acute traumatic brain injury (TBI; reviewedin [118]). Postmortem studies on patients who suffered TBIshow that APP rapidly accumulates together with Ab indamaged axons after trauma, [119] followed by release ofAb into tissue and amyloid plaque formation [120–122]. The presence of Ab plaques after TBI has also beenverified in studies on fresh brain tissue samples excisedduring surgery [123]. A recent PET study also showedincreased cortical PiB binding following severe TBI[124]. It is difficult to perform lumbar puncture after severeTBI, but studies on ventricular CSF samples show amarked increase in Ab42, up to ten times of baseline levels,during the days following trauma [125].

Concluding remarksThe amyloid biomarkers CSF Ab42 and amyloid PET bothshow a high diagnostic ability to identify AD, also duringthe earlier stages of the disease. These biomarkers alsoshow a high concordance, with approximately 90% of casesbeing either positive or negative for both biomarkers. Bothamyloid biomarkers have a central position in the newlyrevised criteria for prodromal AD outlined by Dubois andcoworkers in the International Working Group (IWG) forthe diagnosis of AD [126] and in the National Institute ofAging and Alzheimer’s Association criteria for dementiadue to AD [127] and MCI due to AD [128]. The highconcordance for the amyloid biomarkers suggests that theymay be used interchangeably to aid clinical diagnosiswork-up or patient enrichment in clinical trials. As anexample, several clinical trials, such as the Phase IIIsolanezumab and gantenerumab trials, are using eitherCSF Ab42 or amyloid PET for enrichment of prodromal ADcases in the screening of MCI cases.

Thus, the choice between amyloid PET and CSF bio-markers as diagnostic tools in the clinic will not depend ondifferent performance of these biomarkers to identify AD,but on other factors, such availability, adverse effects,training status, and willingness among clinicians to per-form lumbar puncture, availability of and distance to PETscanners and cyclotrons, and finally financial consider-ations that payers have to make (Table 3). Health economicstudies have shown that the use of CSF biomarkers to

Table 3. Amyloid biomarkers: implementation in the clinica

Feature Amyloid PET CSF Ab42

Invasiveness Radioactive molecule introduced into bloodstream by

intravenous injection

Lumbar puncture, with introduction of needle into

subarachnoid space, performed for CSF collection

Injected PET ligand crosses blood–brain barrier and

binds to amyloid deposits in brain

Total radiation exposure per examination is �9 mSv

when CT scan is performed for reconstruction of

imagesb (for comparison, annual background radiation

in USA is � 3.1 mSv)c

Availability PET instrument needed at hospital Physicians need experience to perform, or training to

start with, CSF collection by lumbar puncture.

Cyclotron needed nearby, because half-life of 18F ligands

is 110 minb

Laboratory need strict internal quality control

systems to assure reproducible results, to both verify

individual runs and maintain long-term stability of

measurements [68]

Adverse effects Headache is most common adverse event (2%)b Headache is most common adverse event (1–3%)

[130–133], but some studies report higher incidence

[134]

Other adverse effects: claustrophobia, anxiety, dizziness,

neck pain, nausea, and fatigueb

Other adverse effects: back pain, dizziness, and

nausea

Interpretation

and reports

PET images labeled as positive or negative by visual

evaluation, by comparing radioactivity in cortical gray

matter with that in adjacent white matter

Reports on CSF Ab42 given as absolute levels, most

often as pg/mL (= ng/L)

Each laboratory has to establish a cut-off level,

because absolute levels vary between laboratories

[76]

Health economics

(price per clinical

biomarker analysis)

Cost of PET scan is s2000–2500 (US$2300–2850) in

Europe and US$3000–4500 in the USA

Cost of lumbar puncture in Europe is estimated at

s150 (US$180) [129], but varies between countries,

and is integrated in general budget for clinics in some

countries (i.e., no extra charge)

Cost of CSF Ab42 test is s35–60 (US$40–70) in Europe

aAbbreviations: CT, computerized tomography.

bExamples of radiation and adverse effects based on Amyvid (http://pi.lilly.com/us/amyvid-uspi.pdf).

cSource for background radiation: http://www.epa.gov/radiation/understand/perspective.html.



identify AD among MCI patients is cost-effective, eventoday when only symptomatic treatments are available[129]. Except for financial considerations, the risk of com-plications after lumbar puncture is a common theme whendiscussing implementation of the amyloid biomarkers inthe clinic. Several studies have showed that the main

Cut-off <192 pg/mL(A)

50

CN AD sMCI pMCI

Low

Nor

mal

100

150

CSF

Ab42

(ng/

L)

200

250

300

350

Figure 3. Raw values for cerebrospinal fluid (CSF) and positron emission tomography (P

the AlzBio3 Luminex assay) and (B) florbetapir amyloid PET as global standardized upt

disease (AD), with a CSF Ab42 level below 192 pg/mL and a global florbetapir SUVR ab

controls; AD, AD dementia; sMCI, stable mild cognitive impairment; pMCI, progressive

study. Reprinted from [74].

complication after LP, post-lumbar puncture headache,is usually mild and of short duration (one or a few days),and has an incidence of 1–3% [130–133], which is in thesame range as the risk of headache after amyloid PET(Table 3), although some studies report a higher frequency[134]. Furthermore, to be implemented in clinical routine

(B)

0.8

1.0

1.2

1.4

1.6

1.8

2.0

CN AD sMCI pMCI

Neg

a�ve

Posi�

ve

PET

Flor

beta

pir (

SUVr

)

Cut-off >1.11 SUVR


ET) amyloid biomarkers. (A) CSF b-amyloid (Ab) 42 levels in pg/mL (analyzed using

ake value ratio (SUVR). Lines represent established cut-offs cutoffs for Alzheimer’s

ove 1.11 (normalized to whole cerebellum) indicative of AD. Diagnostic groups: C,

MCI. Based on data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI)

9

http://pi.lilly.com/us/amyvid-uspi.pdf

http://www.epa.gov/radiation/understand/perspective.html



diagnostics and in clinical trials, standardization effortsare needed both for CSF Ab42 measurements and amyloidPET measurements. This is especially important to enablethese amyloid biomarkers to be used interchangeably intrials, and to allow accurate comparisons between labora-tories and in longitudinal follow-up of patients. It should benoted that, while CSF Ab42 values in clinical routine arereported as absolute levels in pg/mL as compared with acut-off value, amyloid PET scans are reported as eitherpositive or negative (Table 3). However, for both CSF Ab42and global amyloid PET SUVR values, there is a continu-um of values and an overlap between controls and patientswith AD or MCI, without any distinct cut-off (Figure 3);thus, values that are close to the cut-off should be inter-preted with caution.

Except for use in clinical diagnosis and treatment trials,amyloid biomarkers have also proven valuable for under-standing AD pathogenesis and learning about the timecourse and interplay of the different pathogenic mecha-nisms in AD. The highly cited hypothetical model on thetemporal evolution of AD biomarkers (and pathogenesis)was built on clinical biomarker studies [135]. In theupdated model, it is hypothesized that an incident Ab

deposition precedes tau pathology and neurodegeneration[136]. Interestingly, a large report from the DominantlyInherited Alzheimer’s Network (DIAN) study suggestedthat mutation carriers show a decrease in CSF Ab4225 years before expected symptom onset, while amyloiddeposition measured by PET could be detected 15 yearsbefore symptoms, at the same time when CSF levels of tauprotein increase and brain atrophy evaluated by MRI couldbe noted [137]. Longitudinal clinical biomarkers studiesusing novel biomarker modalities, such as tau PET [138]and CSF biomarkers, for synaptic function and degenera-tion, including the presynaptic protein Synaptosomal-as-sociated protein 25 (SNAP-25) [139] and the dendriticprotein neurogranin [140], will add to the understandingof the evolution of pathogenic processes during the courseof AD.

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http://dx.doi.org/10.1016/j.jalz.2014.10.009